1. Field of the Invention
This invention relates to a method for the quantitative determination of free fatty acids (abbreviated throughout the specification and claims as FFA) using an enzyme. The term "free fatty acids" or "FFA" used herein is intended to include any fatty acid whose carboxyl group is not covalently bonded to another compound.
2. Description of the Prior Art
Conventional methods for assaying FFA include the Dore's method wherein FFA is extracted into an organic solvent and titrated with a dilute alkali, the Itaya's method wherein metal salts of FFA are extracted into an appropriate solvent and assayed colorimetrically, and the like. These methods, however, were difficult to standardize because of complicated procedures involved in the extraction with an organic solvent and other problems.
A method for the assay of FFA using an enzyme is reported by Takahashi et al. in Rinsho Kagaku Vol. 4, No. 2, pages 179-185 (1975), which method employs acyl-CoA synthetase enzyme (E.C. 6.2.1.3; hereinafter abbreviated as ACS). Use of enzymes in assay systems has various advantages including the fact that the reactions involved are specific and proceed under mild conditions, and in recent years such enzymatic methods have found particularly wide applications. However, these prior art assay methods using enzymes were difficult to apply to assay for FFA in a system which contains in addition to FFA a protein, particularly such a protein as albumin having a strong affinity for FFA, for example, to assay for serum FFA, since FFA is firmly bound to albumin in such a system and is not readily set free under mild conditions as employed in practice of enzymatic reactions. The nature of the linkage between FFA and albumin is not exactly understood, although it is said to be a hydrophobic bond or an ionic bond.